Streptococcus pyogenes is a Gram-positive bacterium that is the etiological agent of several diseases in humans, including pharyngitis and/or tonsillitis, skin infections (impetigo, erysipelas, and other forms of pyoderma), acute rheumatic fever (ARF), scarlet fever (SF), poststreptococcal glomerulonephritis (PSGN), and a toxic-shock-like syndrome (TSLS). On a global basis, ARF is the most common cause of pediatric heart disease. For example, it is estimated that in India more than six million school-aged children suffer from rheumatic heart disease. In the United States, "sore throat" is the third most common reason for physician office visits and S. pyogenes is recovered from about 30% of children with this complaint. There are about 25-35 million cases of streptococcal pharyngitis per year in the United States, responsible for about 1-2 billion dollars per year in health care costs.
In recent years, an intercontinental increase in streptococcal disease frequency and severity has occurred for unknown reasons, although two variant pyrogenic exotoxin A (SPEA) molecules have been implicated. The amino acid residues characterizing the mutant SPEA molecules are located in an area of the toxin that, based on the recently published three-dimensional crystal structure of the related enterotoxin B from Staphylococcus aureus, form the T-cell receptor binding groove.
S. pyogenes synthesizes an extracellular zymogen of 371 amino acids (40,314 kDa) that can be transformed into an enzymatically active protease of 253 amino acids (27,588 kDa) by autocatalytic conversion. The zymogen contains one or more epitopes not associated with the truncated enzyme. Both the zymogen and active protease contain a single half-cysteine per molecule that is susceptible to sulfhydryl antagonists. In broth cultures, inactive precursor accumulates extracellularly during bacterial multiplication and reaches a maximum concentration at the end of logarithmic growth. Some strains yield up to 150 mg/liter of zymogen, and the molecule is a major extracellular protein. Thus, the streptococcal cysteine protease resembles many secreted bacterial extracellular protease virulence factors in having a specific signal peptide and a pro-sequence that is removed in an autocatalytic fashion to generate a fully active enzyme.
Detection of Group A Streptococcal infections has been hampered by the fact that currently available assays have relied upon detection of the Group A antigen. The extracellular protease, and antibodies generated against it, could also provide the basis of screening assays. Likewise, PCR-based assays could be used for detection of nucleic acid sequences which encode the extracellular protease. It therefore is of interest to identify conserved epitopes, particularly immunodominants conserved epitopes in the cysteine protease molecule, for the development of compositions and methods which can be used for screening for Group A Streptococcus organisms.
The continued great morbidity and mortality caused by S. pyogenes in developing nations, the significant health care financial burden attributable to Group A Streptococci in the United States, and increasing levels of antibiotic resistance in this pathogen highlight the need for a fuller understanding of the molecular pathogenesis of Streptococcal infection. Moreover, the recent disease increase underscores the lack of an efficacious vaccine, despite the repeated inclusion of S. pyogenes in lists of important human pathogens for which vaccines are needed.
Protection against systemic Streptococcal infection is thought to be due predominantly to type-specific opsonic anti-M protein IgG. As a consequence, immunoprophylaxis research has been conducted almost exclusively in the context of formulating an M protein vaccine. However, two major theoretical and practical problems have hindered this approach. First, more than 100 distinctive M protein types have been described based on serological and gene sequencing studies. The occurrence of this extensive array of serotypic variants means either that an effective M protein vaccine must be heterogenous in composition or that conserved protective M protein elements must be used. Formulation of a highly polyvalent vaccine has generated little enthusiasm, and a conserved pan-protective M protein fragment has yet to be identified. A second problem that has plagued M protein vaccine research is the observation that M proteins contain epitopes that cross-react with heart and other human tissue. This fact, in concert with the presumed autoimmune aspects of several Streptococcal diseases, has also slowed M protein vaccine development. It therefore would be of interest to develop an effective vaccine for the immune prophylactic prevention of Group A Streptococcal infection based upon other than the M protein and an efficient method of immunizing a human against Group A streptococcal infections.